The four mammalian Argonaute family members are thought to share redundant functions in the microRNA pathway, yet only AG02 possesses the catalytic slicer function required for RNA interference. Whether AG01, AG03, or AG04 possess specialized functions remains unclear. Given its high expression in mouse spermatocytes, we asked whether AG04 plays a role in meiotic progression. AG04 localizes to spermatocyte nuclei during meiotic prophase 1, specifically at sites of asynapsis and in the transcriptionally silenced XY sub-domain, the sex body, a result that is exciting given the usually cytoplasmic location ofthe Argonaute proteins. We generated Ago4 knockout mice and showed that Ago4-/- spermatogonia initiate meiosis early, resulting from premature induction of retinoic acid-response genes. During prophase I, the sex body assembles incorrectly in Ago4-/- mice, leading to disrupted meiotic sex chromosome inactivation (MSCI) associated with a dramatic loss of microRNAs from meiotic cells. Loss of MSCI results in apoptosis due to expression of Y-linked genes that are toxic to spermatocytes. However, expression analysis reveals that AG03 may at least partially compensate for the loss of Ago4, leading us to hypothesize thatAG03 and AG04 co-operate to regulate key events In mammalian meiosis. The goal of these studies is to further elucidate these roles in meiotic initiation and in meiotic silencing, and to understand further the redundancy that exists between these two Argonautes in these processes. The three specific aims are: (1) To generate and characterize the germ cell phenotype of Ago3.Ago4 mutant mice and to compare germ cell phenotypes between single and double mutant animals, (2) To explore the roles of AG03 and AG04 in meiotic initiation, and (3) To elucidate the role of Argonautes and their RNA cargoes in MSCI and in other meiotic silencing events including meiotic silencing of unsynapsed chromatin (MSUC). Taken together, these studies will provide the first fully integrated view of how different Argonautes function in the testis to promote proper meiotic entry and to ensure meiotic silencing during prophase 1.